DelPrincipe F, Egger M, Ellis-Davies G C, Niggli E
Department of Physiology, University of Bern, Switzerland.
Cell Calcium. 1999 Jan;25(1):85-91. doi: 10.1054/ceca.1998.0009.
We report efficient two-photon and UV-laser flash photolysis of dimethoxynitrophenyl-EGTA-4 (DMNPE-4), a newly-developed photolabile Ca(2+)-specific chelator. This compound exhibits good two-photon absorption at 705 nm, has a low Mg2+ affinity (approximately 7 mM), a Kd for Ca2+ of 19 nM, a quantum yield of 0.20 and changes its Ca2+ affinity by 21,000-fold upon photolysis. Two-photon excitation photolysis (TPP) experiments were performed with a Ti:Sapphire laser in solutions containing DMNPE-4 with either 0 or 10 mM Mg2+ and compared to that of the widely used Ca2+ cage, DM-nitrophen (Kd for Ca2+ 5 nM, Kd for Mg2+ 2.5 microM, quantum yield 0.18, affinity change 600,000-fold). The resulting Ca2+ signals were recorded with the fluorescent Ca2+ indicator fluo-3 and a laser-scanning confocal microscope in the line-scan mode. In vitro, photolysis of DMNPE-4:Ca2+ produced Ca(2+)-release signals that had comparable amplitudes and time courses in the presence and absence of Mg2+. However, photorelease of Ca2+ from DM-nitrophen was obviated by the presence of Mg2+. In patch-clamped isolated cardiac myocytes, equivalent TPP results were obtained in analogous experiments. Single-photon excitation of DMNPE-4 by Nd:YAG laser flashes produced Na-Ca exchange currents of comparable amplitude in the absence and presence of Mg2+. However, only very small currents were observed in DM-nitrophen solution containing 10 mM Mg2+. In conclusion, both DMNPE-4 and DM-nitrophen undergo TPP, however, only DMNPE-4 exhibits efficient release of Ca2+ in the presence of Mg2+.
我们报道了二甲基氧基硝基苯基 - EGTA - 4（DMNPE - 4）的高效双光子和紫外激光闪光光解，DMNPE - 4是一种新开发的对光不稳定的Ca(2+)特异性螯合剂。该化合物在705 nm处表现出良好的双光子吸收，对Mg2+的亲和力低（约7 mM），Ca2+的解离常数（Kd）为19 nM，量子产率为0.20，光解后其Ca2+亲和力变化21000倍。在含有0或10 mM Mg2+的DMNPE - 4溶液中，用钛宝石激光器进行双光子激发光解（TPP）实验，并与广泛使用的Ca2+笼状化合物DM - 硝基酚（Ca2+的Kd为5 nM，Mg2+的Kd为2.5 microM，量子产率0.18，亲和力变化600000倍）进行比较。用荧光Ca2+指示剂fluo - 3和激光扫描共聚焦显微镜在线扫描模式下记录产生的Ca2+信号。在体外，DMNPE - 4:Ca2+的光解在有和没有Mg2+存在的情况下产生了具有可比幅度和时间进程的Ca(2+)释放信号。然而，Mg2+的存在消除了DM - 硝基酚中Ca2+的光释放。在膜片钳分离的心肌细胞中，类似实验获得了等效的TPP结果。用Nd:YAG激光闪光对DMNPE - 4进行单光子激发，在有和没有Mg2+存在的情况下产生了幅度可比的钠钙交换电流。然而，在含有10 mM Mg2+的DM - 硝基酚溶液中仅观察到非常小的电流。总之，DMNPE - 4和DM - 硝基酚都能进行TPP，然而，只有DMNPE - 4在有Mg2+存在的情况下表现出高效的Ca2+释放。
